Tepp 46

ROS production was measured by luminol-dependent chemiluminescence (CL) assay as described by Kanashiro and collaborators^{51 (link)}. Briefly, neutrophils (0.2  × 10⁶) were pre-treated (when indicated) for 1 h with oxalate (3 mM, Sigma, 71800), TEPP-46 (30 µM, Millipore, 5054870001), 2-Deoxy-D-glucose (2-DG, 3 mM, Sigma, D8375), 3PO (10 µM, Calbiochem, 525330), phosphoenolpyruvate (PEP, 1 mM, Sigma, P0564), 5-pentadecylresorcinol (5-PDR, 30 µM, Sigma, 91822) or propranolol (30 µM, Sigma, P0884). Neutrophils were then activated with Zy/op (100 µg/mL) or S. aureus (MOI = 3) in the presence of luminol (Sigma, A8511) 10⁻⁴M or lucigenin (Sigma, M8010). The reaction was monitored in a luminometer (FlexStation 3 Multi-Mode Microplate Reader, Molecular Devices, San Jose, CA) for 1 h, at 37 °C, and the results were expressed as the area under the time-course CL curve (AUC).

Human pancreatic cancer cell lines used in the study are: Capan1, adenocarcinoma cells derived from pancreatic metastatic site, #ATCC HTB-79; Panc1, a pancreatic epitheloid carcinoma cell line, #ATCC CRL-1469; BxPC3, pancreas adenocarcinoma cells, #ATCC CRL-1687 and Mia Paca-2 carcinoma cells, #ATCC CRL-1420. PaTu2 and PancTu1 pancreatic adenocarcinoma cells were kindly provided by Prof. Simone Fulda, Institute for Experimental Cancer Research in Pediatrics, Frankfurt, Germany. BxPC3 and Capan1 were used for in vivo investigations due to their ability to form tumors. Due to higher transient transfection efficacy, PaTu2 and Capan1 were involved in reporter assays and ELISA. Cell lines of early passages were cultured in DMEM (Invitrogen, Germany) supplemented with 10 % fetal calf serum (FCS: Biochrom / Millipore, Germany), 1 % penicillin/streptomycin. BAY 87-2243 was purchased from Seleckchem (#S7309), TEPP-46 was from Millipore (#5.05 487.0001).